This work has two interrelated objectives: (1) To understand the enzymology and regulation of DNA precursor biosynthesis and utilization in animal cells; and (2) To elucidate the biological actions of antimetabolites, notably the fluorinated pyrimidines and the folate antagonists, which exert their primary effects through inhibition of DNA precursor synthesis. On one hand antimetabolites are to be used as probes for exploring intracellular distribution of nucleotide pools, and on the other hand studies on DNA precursor pool kinetics are expected to help clarify the biological effects of the antimetabolites. The project consists of four distinct but related studies: (1) development of new or improved methodology for analyzing nucleotide pools, their labeling kinetics, and their subcellular distributions; (2) Determination of the nature of DNA precursor compartmentation within the cell, both structural and functional; (3) Analysis of nucleotide pools, residual DNA synthesis, and intracellular distribution of folate coenzymes in cells treated with methotrexate or 5-fluorodeoxyuridine; and (4) Studies on the structure and biological functions of organized complexes of enzymes of DNA precursor synthesis, and their relation to the DNA replication apparatus.